Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin

The administration of recombinant methionyl bovine somatotropin (rMbST) to dairy cows to increase milk yield remains a common practice in many countries including the USA, Brazil, Mexico, South Africa and Korea, whereas it has been forbidden within the European Union (EU) since 1999. A rapid screening immunoanalytical method capable of the unequivocal determination of rMbST in milk would be highly desirable in order to effectively monitor compliance with the EU-wide ban for home-made or imported dairy products. For decades, the production of specific antibodies for this recombinant isoform of bovine somatotropin (bST) has remained elusive, due to the high degree of sequence homology between both counterparts (e.g. methionine for rMbST in substitution of alanine in bST at the N-terminus). In this study, we compared several immunizing strategies for the production of specific polyclonal antibodies (pAbs), based on the use of the full-length recombinant protein, an rMbST N-terminus peptide fragment and a multiple antigen peptide (MAP) which consists of an oligomeric branching lysine core attached to the first two N-terminus amino acids of rMbST, methionine and phenylalanine (MF-MAP). The immunization with KLH-conjugated MF-MAP led to the production of the pAb with the highest rMbST/bST recognition ratio amongst the generated battery of antibodies. The pAb exhibited a specific binding ability to rMbST in a competitive antigen-coated ELISA format, which avidity was further improved after purification by rMbST N-terminus peptide-based affinity chromatography. These results suggest that immunodiscrimination between structurally related proteins can be achieved using immuno-enhanced immunogens such as MAPs. © 2012 Elsevier B.V.

Welfare risk assessment:The benefits and common pitfalls

Risk is defined as a situation involving exposure to danger. Risk assessment by nature characterises the probability of a negative event occurring and quantifies the consequences of such an event. Risk assessment is increasingly being used in the field of animal welfare as a means of drawing comparisons between multiple welfare problems within and between species and identifying those that should be prioritised by policy-makers, either because they affect a large proportion of the population or because they have particularly severe consequences for those affected. The assessment of risk is typically based on three fundamental factors: intensity of consequences, duration affected by consequences and prevalence. However, it has been recognised that these factors alone do not give a complete picture of a hazard and its associated consequences. Rather, to get a complete picture, it is important to also consider information about the hazard itself: probability of exposure to the hazard and duration of exposure to the hazard. The method has been applied to a variety of farmed species (eg poultry, dairy cows, farmed fish), investigating housing, husbandry and slaughter procedures, as well as companion animals, where it has been used to compare inherited defects in pedigree dogs and horses. To what extent can we trust current risk assessment methods to get the priorities straight? How should we interpret the results produced by such assessments? Here, the potential difficulties and pitfalls of the welfare risk assessment method will be discussed: (i) the assumption that welfare hazards are independent; (ii) the problem of quantifying the model parameters; and (iii) assessing and incorporating variability and uncertainty into welfare risk assessments. © 2012 Universities Federation for Animal Welfare.

Behavioural and physiological responses of heifer calves to acute stressors: long-term consistency and relationship with adult reactivity to milking

The present study investigated the long-term consistency of individual differences in dairy cattles’ responses in tests of behavioural and hypothalamo–pituitary–adrenocortical (HPA) axis reactivity, as well as the relationship between responsiveness in behavioural tests and the reaction to first milking. Two cohorts of heifer calves, Cohorts 1 (N = 25) and 2 (N = 16), respectively, were examined longitudinally from the rearing period until adulthood. Cohort 1 heifers were subjected to open field (OF), novel object (NO), restraint, and response to a human tests at 7 months of age, and were again observed in an OF test during first pregnancy between 22 and 24 months of age. Subsequently, inhibition of milk ejection and stepping and kicking behaviours were recorded in Cohort 1 heifers during their first machine milking. Cohort 2 heifers were individually subjected to OF and NO tests as well as two HPA axis reactivity tests (determining ACTH and/or cortisol response profiles after administration of exogenous CRH and ACTH, respectively) at 6 months of age and during first lactation at approximately 29 months of age. Principal component analysis (PCA) was used to condense correlated response measures (to behavioural tests and to milking) within ages into independent dimensions underlying heifers’ reactivity. Heifers demonstrated consistent individual differences in locomotion and vocalisation during an OF test from rearing to first pregnancy (Cohort 1) or first lactation (Cohort 2). Individual differences in struggling in a restraint test at 7 months of age reliably predicted those in OF locomotion during first pregnancy in Cohort 1 heifers. Cohort 2 animals with high cortisol responses to OF and NO tests and high avoidance of the novel object at 6 months of age also exhibited enhanced cortisol responses to OF and NO tests at 29 months of age. Measures of HPA axis reactivity, locomotion, vocalisation and adrenocortical and behavioural responses to novelty were largely uncorrelated, supporting the idea that stress responsiveness in dairy cows is mediated by multiple independent underlying traits. Inhibition of milk ejection and stepping and kicking behaviours during first machine milking were not related to earlier struggling during restraint, locomotor responses to OF and NO tests, or the behavioural interaction with a novel object. Heifers with high rates of OF and NO vocalisation and short latencies to first contact with the human at 7 months of age exhibited better milk ejection during first machine milking. This suggests that low underlying sociality might be implicated in the inhibition of milk ejection at the beginning of lactation in heifers.

Calcium binding proteins in the liver fluke, Fasciola hepatica

The common liver fluke, Fasciola hepatica, is a parasite of mammals. In the western world its effects are largely felt on agriculture where infection of cows, sheep and other farm animals is estimated to cause millions of dollars ofif financial losses. In the developing world, the problem is even more serious with an estimated 7 million infected people and many millions more at risk of infection. Calcium signalling is of key importance in all eukaryotic species and recent discoveries of novel types of calcium binding proteins in liver flukes (and related trematodes) suggest that there may be calcium signalling processes which are unique to this group of organisms. If so, these pathways may provide potential targets for the design of novel anthelmintic drugs. Here, we review three main groups of F. hepatica calcium binding proteins: the FH8 family, the calmodulin family (FhCaM1, FhCaM2 and FhCaM3) and the EF-hand/dynein light chain family (FH22, FhCaBP3, FhCaBP4). Considerable information has been gathered on the sequences, predicted structures and biochemical properties of these molecules. The challenge now is to understand their functions in the organism.

Genomic prediction for tuberculosis resistance in dairy cattle

Background: The increasing prevalence of bovine tuberculosis (bTB) in the UK and the limitations of the currently available diagnostic and control methods require the development of complementary approaches to assist in the sustainable control of the disease. One potential approach is the identification of animals that are genetically more resistant to bTB, to enable breeding of animals with enhanced resistance. This paper focuses on prediction of resistance to bTB. We explore estimation of direct genomic estimated breeding values (DGVs) for bTB resistance in UK dairy cattle, using dense SNP chip data, and test these genomic predictions for situations when disease phenotypes are not available on selection candidates. Methodology/Principal Findings: We estimated DGVs using genomic best linear unbiased prediction methodology, and assessed their predictive accuracies with a cross validation procedure and receiver operator characteristic (ROC) curves. Furthermore, these results were compared with theoretical expectations for prediction accuracy and area-under-the-ROC- curve (AUC). The dataset comprised 1151 Holstein-Friesian cows (bTB cases or controls). All individuals (592 cases and 559 controls) were genotyped for 727,252 loci (Illumina Bead Chip). The estimated observed heritability of bTB resistance was 0.23±0.06 (0.34 on the liability scale) and five-fold cross validation, replicated six times, provided a prediction accuracy of 0.33 (95% C.I.: 0.26, 0.40). ROC curves, and the resulting AUC, gave a probability of 0.58, averaged across six replicates, of correctly classifying cows as diseased or as healthy based on SNP chip genotype alone using these data. Conclusions/Significance: These results provide a first step in the investigation of the potential feasibility of genomic selection for bTB resistance using SNP data. Specifically, they demonstrate that genomic selection is possible, even in populations with no pedigree data and on animals lacking bTB phenotypes. However, a larger training population will be required to improve prediction accuracies. © 2014 Tsairidou et al.

Productive Synergies in the City

The chapter focuses on the development of sustainable growing infrastructure in the city at two scales. Firstly the development of a large-scale city wide fuel productive landscape through the development of algae arrays in Liverpool and their connection through urban agriculture systems to develop a closed-cycle food and energy system where waste is food and secondly a hyper-localised neighbourhood food production system in Salford UK that utilises a closed cycle aquaponic system to re-invigorate an urban food desert.

The author develops a three-part model for the implementation of urban agriculture based on hardware (the technological system), software (the biological components) and interface (the links to food and other social networks). The conclusion being that it is possible to develop urban agriculture in cities if their implementation is seen as a process, rather than a static design. Also that as the benefits of such systems are wider than purely the physical outputs of the system in terms of energy and food, and thus we should re-evaluate the purely economic model of appraisal to include these.

Effect of feed space allowance and period of access to food on dairy cow performance

Two experiments were conducted to examine the ‘long-term’ effect of feed space allowance and period of access to feed on dairy cow performance. In Experiment 1, three horizontal feed space allowances (20, 40 and 60 cm cow−1) were examined over a 127-d period (14 cows per treatment). In Experiment 2, 48 dairy cows were used in a continuous design (10-week duration) 2 × 2 factorial design experiment comprising two horizontal feed space allowances (15 and 40 cm cow−1), and two periods of access to feed (unrestricted and restricted). With the former, uneaten feed was removed at 08·00 h, while feeding took place at 09·00 h. With the latter, uneaten feed was removed at 06·00 h, while feeding was delayed until 12·00 h. Mean total dry-matter (DM) intakes were 19·0, 18·7 and 19·3 kg cow−1 d−1 with the 20, 40 and 60 cm cow−1 treatments in Experiment 1, and 18·1 and 18·2 kg cow−1 d−1 with the ‘restricted feeding time’ treatments, and 17·8 and 18·1 kg d−1 with the ‘unrestricted feeding time’ treatments (15 and 40 cm respectively) in Experiment 2. None of milk yield, milk composition, or end-of-study live weight or condition score were significantly affected by treatment in either experiment (P > 0·05), while fat + protein yield was reduced with the 15-cm treatment in Experiment 2 (P < 0·05). When access to feed was restricted by space or time constraints, cows modified their time budgets and increased their rates of intake.

An optimised milk testing protocol to ensure accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis by the PMS-phage assay

There is interest in determining levels of Mycobacterium avium subsp. paratuberculosis (MAP) contamination in milk. The optimal sample preparation for raw cows' milk to ensure accurate enumeration of viable MAP by the peptide-mediated magnetic separation (PMS)-phage assay was determined. Results indicated that milk samples should be refrigerated at 4 C after collection and MAP testing should commence within 24 h, or samples can be frozen at 70 C for up to one month without loss of MAP viability. Use of Bronopol is not advised as MAP viability is affected. The vast majority (>95%) of MAP in raw milk sedimented to the pellet upon centrifugation at 2500 g for 15 min, so this milk fraction should be tested. De-clumping of MAP cells was most effectively achieved by ultrasonication of the resuspended milk pellet on ice in a sonicator bath at 37 kHz for 4 min in ‘Pulse’ mode.

Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia-Romagna Region (Italy) by qPCR

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows’ milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia-Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900-qPCR. Seven hundred and eighty-nine samples tested MAP-positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL−1 (and maximum load of 1424 MAP cells mL−1). This study has shown that a small proportion (3.1%) of BTM samples from Emilia-Romagna region contained MAP in excess of the limit of detection (1.5 × 101 MAP cells mL−1), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production.

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk

Background: Nicotinamide riboside (NR) is a recently discovered NAD+ precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD+ precursors was unknown.

Objective: We aimed to determine NAD+ precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk.

Methods: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD+ precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk.

Results: Cow milk typically contained ∼12 μmol NAD+ precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD+ metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = −0.58, P = 0.014), and NR was degraded by S. aureus. Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk.

Conclusions: NR is a major NAD+ precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD+ precursor vitamin concentration of milk.

Pesquisa de Campylobacter fetus em amostras prepuciais de touros da região do Alentejo

A bactéria Campylobacter fetus divide-se em duas subespécies C. fetus fetus (Cff) e C. fetus venerealis (Cfv). Cfv é o agente patogénico responsável pela campilobacteriose genital bovina (CGB), uma doença que provoca infertilidade em vacas. Cff pode provocar abortos esporádicos, mas não provoca CGB. Neste trabalho procedeu-se à pesquisa de C. fetus por imunofluorescência direta (IFD) em 117 amostras prepuciais, recolhidas por raspagem, de touros pertencentes a 25 explorações na região do Alentejo. A prevalência real de CGB na amostra, considerando a sensibilidade e especificidade do teste, foi de 52,89% de animais e 80% de explorações. Foi também realizada uma comparação entre dois testes de diagnóstico de CGB, a IFD e o isolamento bacteriológico. O isolamento bacteriológico foi difícil de executar e a presença de bactérias contaminantes foi bastante problemática, levando a resultados falso-negativos. A sua correlação com a IFD foi muito baixa, tendo esta revelado ser muito mais sensível; #### Abstract Search for Campylobacter fetus in preputial samples of bulls from Alentejo region The bacteria Campylobacter fetus comprises two subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). Cfv is the pathogen responsible for bovine genital campylobacteriosis (BGC), a disease that causes infertility in cows. Cff may lead to sporadic miscarriages, but does not cause BGC. In this work we searched for C. fetus by direct immunofluorescence (DIF) in 117 preputial samples, collected by scraping, from bulls from Alentejo region. The actual prevalence of BGC, considering the sensitivity and specificity of the test was 52.89% of animals and 80 % of farms. It was also performed a comparison between two BGC diagnostic tests, bacterial isolation and DIF. The bacterial isolation was difficult to perform and the presence of bacterial contaminants was very problematic, leading to false-negative results. The presence of bacterial contaminants in bacterial isolation was problematic, leading to false-negative results. Its correlation with DIF was quite low, and DIF proved to be much more sensitive.

Metais em plantas e algas : acumulação e potencial aplicação em processos de remediação

The present work reports the study of the bioaccumulation of potentially toxic elements (cadmium, lead and mercury) by marine macroalgae (Ulva lactuca, Fucus vesiculosus and Gracilaria gracilis), abundant in the coast and estuarine systems worldwide. These organisms proved to be capable of withstanding moderate multi-metallic contamination (environmentally relevant concentrations), incorporating high amounts of metal in their tissues. The high removal percentages achieved, in particular for mercury (99%), demonstrate the potential of these algae as a basis for a new biotechnological treatment of saline waters contaminated with metals (more efficient, cost-effective and environmentally friendly than conventional methods). U. lactuca was considered the most promising due to the better performance presented. The comparison between the bioaccumulation and biosorption processes suggested that in some cases the use of the living organism will have advantages over the application of biomass, due to the simplicity of the overall process, and the lower residual concentration of metal achieved in the solution (especially for Cd). The transfer and accumulation of Hg by terrestrial plants (Brassica juncea and Lolium perenne) in agricultural fields near a contaminated industrial area was also studied. Despite the low bioaccumulation factors found (<1), there were high Hg content in plants (up to 84 mg kg-1 in roots and up 6.9 mg kg-1 in shoots, dry weight). Daily intake estimates for grazing animals (cows and sheep) pointed to the potential risk to human health derived from consumption of their meat. The results highlighted the important role that plants and algae may have in protection, risk assessment and remediation of environmental systems contaminated with metals.

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed

Background: Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. Methods: 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. Results: Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, Rt = 5.7 ± 1.4, Ho = 0.63 ± 0.19 and He = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. Conclusions: Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.

Identification of descendants of an extinct bovine population from the Algarve region of Portugal using numerical taxonomy analysis of morphological traits

The morphology of a sample of four bulls and 43 cows, presumed to be descendants of the extinct cattle breed ‘Algarvia’ (AG), was used to assign their relationship with animals from other Portuguese autochthonous breeds – Arouquesa (AR), Barrosa˜ (BA), Cachena (CA), Marinhoa (MA), Maronesa (MO), Minhota (MN), Mirandesa (MI), (only bulls), Alentejana (AL), Garvonesa (GA), Mertolenga (ME) and Preta (PR). Standard numerical taxonomic methods were applied to a set of 183 (cows) and 170 (bulls) traits, to derive average pairwise taxonomic distances among the sample of 257 cows and 76 bulls. Distance coefficients (morphological index of distance) ranged from 0.22 to 2.62 (cows) and from 0.49 to 2.13 (bulls). Unweighted pair group method using arithmetic averages (UPGMA)-based phenograms and a principal coordinate analysis showed that bulls were highly clustered and cows showed a tendency to cluster according to their geographical and breed origin. The AG population grouped together with GA, AL, ME and MN breeds in the Red Convex group. The average taxonomic distance among breeds was 1.02, the highest being 1.39 (ME versus BA) and the lowest being 0.64 (MA versus AR). The approach allowed for the identification of a phenotypically differentiated set of animals, comprising 19 cows and four bulls representative of the AG breed, and which can be targeted in further studies aiming at the recovery of this extinct breed.

Analyse de la fertilité des vaches laitières Holstein «Repeat Breeder»

L’importance de la problématique des vaches laitières non gestantes avec multiples inséminations (› 3) communément appelées « Repeat Breeder » (RB), au Québec, repose sur deux facteurs: son incidence et son impact économique. Actuellement, l’incidence du syndrome RB au Québec est de ± 25% (Rapport annuel, juin 2008, www.dsahr.ca). Les pertes monétaires associées à ce problème proviennent des frais vétérinaires et d’insémination, de la diminution de productivité ainsi que des pertes en lien avec la réforme involontaire. Afin d’avoir de meilleures connaissances sur ce syndrome, nous devons connaître les facteurs de risques généraux impliqués et ensuite explorer la condition individuelle de ces vaches problèmes. Dans la première partie de ce mémoire, une banque de données informatisées répertoriant 418 383 lactations fut analysée. L’objectif de ce projet de recherche était d’évaluer l’impact des problèmes reproducteurs post-partum et l’effet du nombre de lactations comme facteurs de risques de la vache Repeat Breeder. L’analyse a permis d’établir la dystocie comme étant la condition ayant le plus de conséquences sur la fertilité future. D’autres facteurs de risques à savoir le nombre de lactations influencent le pronostic reproducteur. La deuxième partie de ce mémoire consistait à explorer, à l’aide d’outils cliniques, la condition individuelle des vaches RB. Une étude cohorte fut menée sur des vaches Holstein en fin de période d’attente volontaire au jour 7 du cycle oestral. Les tests cliniques étudiés furent la vaginoscopie, l’examen transrectal, l’ultrasonographie du système reproducteur, la présence d’estérases leucocytaires, la bactériologie et la biochimie du liquide utérin, la cytologie endométriale et le profil de progestérone sérique. Les résultats de ces tests cliniques dévoilent que l’examen bactériologique du liquide utérin est révélateur du statut reproducteur futur.